In Vitro and In Vivo Studies of the Trypanocidal Effect of Novel Quinolines

Compounds.The synthesis and characterization of the 10 quinolines (Table 1) can be found in the supplemental material. For T. cruzi assays, Bz (2-nitroimidazole; Laboratório Farmacêutico do Estado de Pernambuco [LAFEPE], Brazil) was used as a reference drug, and stock solutions were prepared in dimethyl sulfoxide (DMSO), with the final concentrations of the solvent never exceeding 0.6% and 10% in assays in vitro and in vivo, respectively, which are not toxic to the parasite, mammalian cells, and mice. For T. b. rhodesiense, pentamidine and melarsoprol were used as reference drugs. Pentamidine (Sigma) was dissolved in DMSO, and melarsoprol (Arsobal; Aventis) was dissolved in water.

Computational assessment of drug-like properties.Absorption, distribution, metabolism, excretion, and toxicity (ADMET and Lipinski's rule of five) properties of the studied quinolines were evaluated by using the pKCSM approach, which uses graph-based signatures to develop predictive ADMET (22, 24).

Parasites. (i) T. cruzi.BT forms of the Y strain were obtained from blood samples of infected albino Swiss mice at the peak of parasitemia. The purified parasites were resuspended in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum as reported previously (25, 26). Trypomastigotes of the Tulahuen strain expressing the Escherichia coli β-galactosidase gene were collected from the supernatant of T. cruzi-infected L929 cultures as reported previously (16, 27).

(ii) T. brucei.T. b. rhodesiense strain STIB900, a derivative of strain STIB704, was isolated from a patient in Ifakara, Tanzania, in 1982 (28). Bloodstream forms were used for in vitro screening as well as for the acute mouse infection model, which mimics the first stage of HAT.

Mammalian cell cultures.For toxicity assays on mammalian cells, primary cultures of cardiac cells (CC) obtained from mouse embryos were plated onto 96-well plates previously coated with 0.01% gelatin (25). L929 cell lineages were obtained as described previously by Romanha et al. (16). L6 cells (rat skeletal myoblast; ATCC CRL-1458) were maintained in RPMI 1640 medium supplemented with 2 mM l-glutamine, 5.95 g/liter HEPES, 2 g/liter NaHCO₃, and 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO₂ (29).

In vitro cytotoxicity tests.CC were incubated for 24 h at 37°C with different concentrations of each compound (up to 400 μM) diluted in DMEM; the morphology, cell density, and spontaneous contractibility were then evaluated by light microscopy; and cellular viability was determined by the PrestoBlue test as reported previously (27). L929 cells were incubated for 96 h at 37°C with different concentrations of each compound (up to 96 μM) diluted in RPMI, and cellular viability was determined by the alamarBlue test as reported previously (27). The results were expressed according to the manufacturer's instructions, and the LC₅₀ value, which corresponds to the concentration that reduces cellular viability by 50%, was determined. Cytotoxicity assays performed by using L6 cells were conducted with a 72-h compound exposure time as previously reported (29). The selectivity index (SI) was expressed as the ratio between the LC₅₀ values obtained on host or L6 cells and the EC₅₀ obtained for the parasites.

Trypanocidal activity.For T. cruzi assays, BT of the Y strain (DTU II) (30) (5 × 10⁶ BT per ml) were incubated for 2 and 24 h at 37°C in RPMI in the presence or absence of serial dilutions of the compounds (up to 32 μM). After compound incubation, the death rates of parasites were determined by light microscopy through the direct quantification of the number of live parasites by using a Neubauer chamber, and the EC₅₀ (the compound concentration that reduces the number of parasites by 50%) was calculated (22). For assaying intracellular forms of the Y strain (DTU II), the most promising compound was further evaluated with infection of primary cultures of CC (using a ratio of 10 BT to 1 host cell). After 24 h of parasite interaction, the cultures were rinsed and incubated with the compounds for 48 h. After fixation with Bouin solution and staining with Giemsa solution, the percentage of CC infection and the mean number of parasites per infected cell were calculated by light microscopy for the determination of EC₅₀s of the infection index (II) (II = % infected host cells × mean number of parasites per cell) (26). Culture-derived trypomastigotes of T. cruzi (Tulahuen strain expressing β-galactosidase [DTU VI]) were used to infect L929 cultures at a ratio of 10:1 (parasites/host cell). After 2 h, the cultures were washed and cultivated for another 48 h for the establishment of infection. The compounds were then added at increasing nontoxic concentrations to mammalian host cells, followed by maintenance at 37°C for 96 h for the determination of EC₅₀s. After the addition of 50 μl of the substrate (CPRG [chlorophenol red glycoside] at 500 mM) in 0.5% Nonidet P40 and incubation at 37°C for 18 h, the absorbance at 570 nm was measured, and results were expressed as percent inhibition of the infection rate (16). For T. brucei assays, bloodstream forms of T. b. rhodesiense strain STIB900 were incubated in minimum essential medium (MEM) for 72 h in the presence of 3-fold serial dilutions at 37°C in a humidified atmosphere containing 5% CO₂. Parasite viability was assessed with the viability marker resazurin after a 3-day drug exposure time as previously reported (29).

Mouse acute toxicity.In order to determine the no-observed-adverse-effect level (NOAEL), increasing doses of the tested compounds (up to 200 mg/kg of body weight) were injected by the i.p. route individually into female Swiss mice (21 to 23 g; n = 2 per assay of the tested compounds). Treated animals were inspected for toxic and subtoxic symptoms according to Organization for Economic Cooperation and Development (OECD) guidelines (33). Forty-eight hours after compound injection, the NOAEL values were determined as reported previously (31). Biochemical analyses performed 48 h after compound exposure were performed at the ICTB platform (Fiocruz/RJ) as reported previously (17, 31).

Mouse infection and treatment.For acute T. cruzi infection models, male and female Swiss Webster mice (18 to 20 g) obtained from the animal facilities of ICTB were housed at a maximum of 7 per cage, kept in a specific-pathogen-free (SPF) room at 20°C to 24°C under a 12-h-light/12-h-dark cycle, and provided with sterilized water and chow ad libitum. The animals were allowed to acclimate for 7 days before the start of the experiments. Infection was performed by i.p. injection of 10⁴ bloodstream trypomastigotes (Y strain). Age-matched noninfected mice were maintained under identical conditions (26). Quinolines were first dissolved in DMSO and then freshly diluted with sterile distilled water. The stock solution of Bz was prepared in sterile distilled water with 3% Tween 80 (Sigma-Aldrich). The animals were divided into the following groups (n > 3 per group): uninfected (noninfected and nontreated), untreated (infected but treated with the vehicle only), and treated (infected and treated with the compounds). Therapy was performed through the administration of 5 to 20 mg/kg at parasitemia onset (5 dpi) and the peak of parasitemia (8 dpi) according to an animal model reported previously (23). Alternatively, the most promising quinoline derivatives were administered for five consecutive days, starting at 5 dpi, using up to 25 mg/kg/day (i.p.) and 100 mg/kg/day Bz (orally [p.o.]). For all assays, only mice with positive parasitemia were used in the infected groups. Parasitemia levels in T. cruzi assays were individually checked by direct microscopic counting of parasites in 5 μl of blood, and mortality rates were checked daily until 30 days posttreatment and expressed as a percentage of cumulative mortality (CM) as described previously (27).

For T. brucei models, efficacy experiments were performed as previously reported (32), with modifications to soften the stringency of the mouse model of infection, and in line with the 3R principles for animal testing (reduce, refine, and replace), the number of mice was reduced in the primary in vivo screen. Female NMRI mice were infected i.p. with 10⁴ T. b. rhodesiense STIB900 bloodstream trypanosomes. Experimental groups of two mice were treated with the new test compounds at 40 mg/kg i.p. on three consecutive days from day 1 to day 3 postinfection (total dose of 120 mg/kg i.p.). A control group was infected but remained untreated. The tail blood of all mice was checked microscopically for parasitemia reduction 24 h and 96 h after the last dose. Parasite reduction in mice treated with the experimental compounds was compared with that in the untreated control mice. Mice were euthanized at 96 h posttreatment if parasites were still detected in the tail blood. Aparasitemic mice were further examined twice per week for 30 days, or mice were euthanized after parasitemia relapses were detected. Mice that remained aparasitemic until day 30 were considered cured. The data from the follow-up efficacy study for compounds that achieved a parasite reduction of at least 98% in one of the two treated mice were comparable to those of groups of 4 infected mice treated for four consecutive days with a higher dosage of 50 mg/kg i.p. (total dose of 200 mg/kg i.p.). Pentamidine was used as a positive drug control, and it cured mice at 3× 4 mg/kg and 4× 1 mg/kg i.p.

Statistical analyses.Statistical analyses were performed by analysis of variance (ANOVA) with the level of significance set at a P value of ≤0.05.

Ethics.All animal procedures performed at Fiocruz were carried out in accordance with the guidelines established by the Committee of Ethics for the Use of Animals (CEUA LW16/14). All protocols and procedures using animal models of T. brucei infection were reviewed and approved by the local veterinary authorities of the Canton Basel-Stadt, Switzerland.